Anal g gesiaxoat t tail intravenous injection



United States Patent Claims. 61. 260-243) This invention relates, ingeneral, to organic chemistry, and involves the provision of novelderivatives of Z-phenylamino-4H-5,6-dihydro-1,3-thiazines. Inparticular, the invention is directed to the provision of certaincompounds of the general class described which are found to possessunusual and outstanding pharmacodynamic activity. The inventionadditionally involves the provision of several alternate syntheses forthe production of these compounds.

It has been known heretofore that one may obtain 2 phenylamino 4H5,6-dihydro-1,3-thiazines, unsubstituted or monosubstituted in thebenzene ring, by reacting aryl isothiocyanates with 3-aminopropanol ortrimethylene imine, and cyclisizing the resulting thioureas by heatingin concentrated hydrochloric acid [M. Tisler, Arch. Pha-rm., 293, 621(1960)]. It is further known that the reaction of 'y-iodopropylaminewith p-tolyl isothiocyanate yields 2 p tolylamino 4H-5,6-dihydro-l,3-thiazine [M. Frankel, Ben, 30, 2497 (1897)]. These known compounds,however, possess little or no pharmacological activity. Thus, the2-o-tolylamino-4H-5,6-dihydro-1,3-thiazine exhibits only a weaktranquilizing activity, for example.

The present invention is based, in part, on the discovery that compoundsof the general class described which possess truly outstandingpharmacological activity are produced when:

(a) Poly-substituted phenyl isothiocyanates are added to3-aminopropanol-( 1) or trimethylene imine to yield the correspondingthioureas which are then treated with acids, or

(b) Poly-substituted phenyl isothiocyanates are reacted with3-halopropylamines, or

(c) N-phenyl-thioureas, poly-substituted on the henzene ring, arereacted with 1,3-dihalopropanes or 3-ha1opropylamine salts, or

(d) aromatic amines, poly-substituted on the benzene ring, are reactedwith 'y-halopropyl isothiocyanates or (e) Aromatic amines,poly-substituted on the benzene ring, are reacted with 2-amino-,2-mercapto-, or 2- alkylmercapto-4H-5,6-dihydro-1,3-thiazines,optionally in the presence of acids.

Typical preferred substituent groups of the foregoingv well as inaliphatic, or aromatic solvents, or in the melt, the particular choiceof a suitable solvent or diluent merely being determined in establishedmanner having reference to the stability and reactivity of the specificreaction components in each instance. In this connection, the use ofsolvents or diluents generally proves to be expedient, but is notabsolutely necessary.

The compounds of the invention which, as pointed out hereinabove areunique in the fact that they possess a surprising variety ofpharmacodynamic activities, are intended for administration astherapeutic agents in base form or in the form of their salts withnon-toxic inorganic or organic acids. Thus, they exhibit an analgeticactivity similar to that of morphine, which renders it possible toperform surgical operative treatments on warm-blooded animals, includingeven abdominal operations, without causing the classical symptoms oftotal anesthesia in the EEG. On the other hand, these derivativesexhibit a strong tranquilizing activity upon peroral administration.Additionally, certain of the novel compounds exhibit a remarkablecoronary activity with respect to increasing the oxygen saturation inthe coronary sinus venous blood on an anaesthesized dog, for example,following intravenous injection, and even at relatively low dosagelevels, whereas, on the other hand, certain of the compounds of theinvention are characterized by strong stimulant effects. Theseactivities have been demonstrated in an extensive series ofpharmacological studies conducted with a typical compound of theinvention (Example I), which shall be described in detail here inafter.

For present purposes, however, it is believed that the invention may bebest understood by reference to the following specific examplesillustrating the application of the foregoing principles and proceduresin the production of typical compounds of the invention:

Example I 2,6-dimethylphenyl isothiocyanate, in amount of 31 grams (0.2mole), prepared from 2,6-dimethylaniline with thiophosgene, were addeddropwise during 15 minutes to a well-stirred suspension of 15 grams (0.2mole) of 3-aminopropanol-(1) in cubic centimeters of ether. The etherstarted to boil. Stirring under reflux was continued for 30 minutes, andthe ether was then distilled off. The residue was treated with 100 cubiccentimeters of concentrated hydrochloric acid and boiled under refluxfor 30 minutes. After cooling, it was diluted with water, filtered freefrom impurities, and the base was precipitated by the addition ofconcentrated sodium hydroxide solution. When recrystallized frombenzene-ligroin, the resulting compound2-(2,6-dimethylphenylamino)-4H-5,6-dihydro-1,3-thiazine, melts at 142 C.The yield amounted to 90% of the theoretical.

Analysis.C I-I N S 1 C H N S Calculated 65. 41 7. 32 12. 72 14. 55 Found65. 64 7. 39 12. 83 14. 12

The same compound was obtained in a yield of 85% when 50 cubiccentimeters of water were employed as the solvent in place of the ether.

@3 With reference to the following structural formula, the compoundslisted in tabulated form below were prepared from the correspondingisothiocyanates and aminopropanols by application of analogoustechniques:

CH N ll CH, NHR

TABLE I Melting R Formula point,

3,4,5-trimethoxyphenyl- 121-123 2-methyl-5-chlorophenylg 1% 14 2o 2 12450mm lphenyl' {CuHzoNgSJlCL 234-236 2,6-diispropylphenyl CmHztNzS151-154 2,4,6-trimethy1phenyl- 122125 2-ethyl-6-methy1phenyl- 85- 882,4-dimethylphenyl- 96- 98 2,3-dichlor0phenyl- 128-1292,5-dichlor0phenyl- 198-200 158-162 2,4,5-tr1chl0ropheny l- 2144152,4,6-trichlorophenyl- CmH@Cl3N2S 169-171 2,3,4,5,6-pentaehlorophenyl-C1UH7C15N2S.HC1 255-258 2,6-diisopropyl-4chlorophenyl Example Il-Bromopropylamine hydrobromide, in amount of 43.8 grams (0.2 mole), weredissolved in a little alcohol and treated in the cold with a solution of0.2 mole of sodium ethoxide. The NaBr was filtered off, 35.5 grams (0.2mole) of 2-ethyl-6-methylphenyl isothiocyanate were added to thefiltrate, and it was boiled under reflux for 5 hours. After the alcoholhad been distilled off, it was dissolved in water, the base wasprecipitated with sodium hydroxide solution, and it was recrystallizedfrom benzene-ligroin. The compound,2-(2-ethyl-6-methylphenylamino)-4H-5,6-dihydro-1,3-thiazine of meltingpoint 85- 88 C. was thus obtained in a yield of 62.5% of thetheoretical.

Example III l C H Cl- Cl N S Calculated- 40. 86 4. 69 11. 35. 74 9. 4110. 77 Found 40. 71 3. 89 11. 98 35. 19 9. 09 11. 05

With reference to the following structural formula, the compoundsindicated in tabulated form below were obtained in an analogous manner:

TABLE II R Formula M.P., Yield,

0. percent 2,6-diisopr0pylphenyl- C1 H24N2S.HOl 254256 412-ehloro-6-methylphenyl- C11H13OlN2S.HC1 182-183 40 Example IV Example VEighteen (18) grams (0.1 mole) of N-(2,6-dimethylphenyl)-thiourea werethoroughly triturated wtih 13.4 grams of 3-chloropropylamine-(1)hydrochloride and melted together. The reaction set in at about 150 C.with a rise in temperature. It was maintained at 150 C. for another 15minutes. The melt was boiled out twice with acetone for the removal ofany possibly unchanged thiourea. The residue was dissolved in verydilute hydrochloric acid and filtered free from impurities. The base wasthen precipitated from the aqueous solution with sodium hydroxidesolution, and was taken up with methylene chloride. When the methylenechloride solution had been dried and the solvent removed,2-(2,6-dirnethylphenylamino)-4H-5,6-dihydrol,3-thiazine was distilled atabout 180 C. and at a pressure of 0.1 mm. When recrystallized frombenzene-ligroin, the compound melts at 140-142 C. It was found to beidentical with the compound described in Example I.

Example VI A mixture of 7.4 grams (0.05 mole) ofZ-methylmercapto-4H-5,6-dihydro-1,3-thiazine and 7.9 grams (0.05 mole)of 2,4-dimethyl-aniline hydrochloride was heated in an oil bath. Theevolution methyl mercaptan strated at about C., and is vigorous at C. Itwas stirred as soon as the mixture had become liquid. It was heated at115-120 C. for 45 minutes, and then at -140 C. for 5 minutes longer. Theresulting melt which is glasslike at room temperature was dissolved inwater, and the base was precipitated with concentrated sodium hydroxidesolution and recrystallized from ligroin. The resulting compound,2-(2,4-dimethylphenylamino)-4H-5,6-dihydro- 1,3-thiazine melts at 9197C. and is identical with the product prepared from 3-aminopropanol and2,4-dimethylphenyl isothiocyanate. The yield was 4.7 grams which isequivalent to 43% of the theoretical.

In general, the compounds of the invention were deemed worthy ofextensive pharmacological investigation by virtue of a distinct bloodpressure-increasing and analgesic activity noted in preliminaryexperiments, whereas further pharmacological analysis revealed thatcertain of the compounds also exhibited considerable ataratic andintensified narcotic effects. Inasmuch as this spectrum of effects is indistinct contrast with known compounds of the same general class, thecompound of Example I, namely, 2-(2,6-dimethylphenylamino)-4H-5,6-dihydro-1,3-thiazine, was selected forintensive pharmacological investigation since it evidenced particularpolyvalency of effects. Thus, this compound is indicated, on a clinicalbasis, for psychiatric use in the treatment of psychotic conditions, inanaesthesiology for premedication, in internal medicine as a hypnoticum,analgesicum and tranquilizer.

Chemically, the compound of Example I is an almost tasteless, colorless,crystalline organic base, insoluble in water and alkalies, soluble indilute acids, and in most or- 5 ganic solvents, including, for example,benzene, acetone, chloroform, etc., but only sparingly soluble inpetroleum ether.

For experimental purposes and ease of identification, throughout thepharmacological investigations, the compound of Example I was designatedby the code designation Wh7286, and in the data presented hereinafter,the same designation will be employed. These data include the followingcategories, in the order indicated:

I. (l) Predominantly central effects:

(a) Single administration (b) Reiterated dispensation (2) Ataracticproperties (3) Action on fishes:

(a) Effect on Betta splendens (b) Effect on guppy (4) Analgeticproperties:

(a) Analgesia test, on mice (tail) (b) Tooth test, on rabbits (c)Pressure test, on cats (tail) ((1) Analgesia test on the inflamed catpaw (e) Joint test, on rats (5) Effect on polysynaptic reflexes:

(a) Cats (b) Rabbits (6) Effect on body temperature:

(a) With cats at the normal temperature (b) Antipyresis in guinea pigs(7) Effect on barbiturate narcosis:

(a) Mice (b) Cats (8) Effect on respiration:

(a) Rabbits (b) Dog (9) Anticonvulsive action in mice:

(a) Electric shock (b) Metrasol cramp (c) Strychnine cramp (d) Nicotinecramp (10) Antitussive action (11) Antagonistic action againstcentral-excitation compounds, in rats:

(a) Tryptamine (b) Apomorphine (c) Methamphetamine (12) Effect on EEG:

(a) Spontaneous EEG (b) EEGArousal reaction (c) Antidote tests NicotineMethamphetamine Lysergic acid diethylamide Physostigmine TryptamineLevallorphan (13) Surgical tests:

(a) With cats (b) With dogs (c) With Rhesus monkeys 6 II. Predominantlyperipheral effects- (14) Dropsy inhibition:

(a) Yeast edema of the cats paw (b) Formalin edema of the cats paw (15)Local anaesthesia:

(a) Conductive anaesthesia (b) Surface anaesthesia (16) Effect onsmooth-muscle organs:

(a) Isolated intestine (b) Sperm vesicle (rat) (c) Uterus (rabbit) 17)Antihistamine effect l8) Antiasthmatic effect (19) Effect on intestinalmobility (20) Diuresis (21) Synaptic effects:

(a) Myoneural transmission (b) Nick-titating membrane (22) Effect oncirculation:

(a) Of wide-awake dogs and cats (b) Of narcotized cats (c) In spinalcats (d) [Peripheral vascular ducts (a) Frog ([3) Guinea pig (v) Rabbitear (6) Dog (23) Cardiac effects:

(a) Guinea pig heart, according to Langendorff (b) 0 content of venousblood (0) Effect on the EKG (24) Effects on blood gases Tolerance- (25)Toxicity with a single dose (26) Toxicity With reiterated doses (27)Local tolerance (28) Further personal tests IV. Summation andRecapitulation III.

Pharmacology I. Predominantly central effects 1) Observations of thebehavior of various animal species:

(a) Single dose (Table I ).Wh7286 has distinct sedative properties,which, with an increased dosage, change over into narcosis-like stateswith strong analgesia.

(b) Repeated d0ses.3 mg./kg. of Wh7286 was administered intravenously to2 cats on each of 5 successive Work days. The typical reactions, viz.interference with the coordination, analgesia, and side turn, did notchange essentially in the course of the test, although the duration ofthe effects appeared to become slightly shorter from day to day. Bothanimals lost weight.

On 12 days 5 mg./kg. of Wh7286 was administered subcutaneously each dayto a monkey. At the start, intensely distinct coordinationinterferences, which after 5 days became slighter. (At the end of thetreatment the total duration of the effects appeared to have becomeshorter.) The analgetic effect, fatigue, and greatly restricted mobilityappeared, however, to be unchanged up to the end of the treatment. Inspite of a strong attenuation -of the appetite there was some foodintake.

TABLE 1.PRESENTATION OF THE ACTION OF Wh7286 Animal Mode of Dose, NumberComportment administration mgJkg. animals of Ferret Subcutaneous. 1.0 2Slackeraing of the movements, coordination maintaine Do 2. 5 2 Totteringgait, animals are motionless. Do 5.0 3 Stuporous, narcotic-likecondition. reaction to painful irritation greatly attenuated. Do 10. O 1Do. 20. 0 1 D0.

TABLE 1-C0ntznued Animal Mode of Dose, Number Comportment administrationmgJkg. animals of R it Intravenous..- 3. -10. 0 3 Flabby, belly and sidepositions well endured,

reacted to a pain-inducing irritation.

Do 10. 0-20. 0 3 Occasional cramps, otherwise as above. From 16 mg.onward, died. Subcutaneous. 5. 0-20. 0 2 Flabbyf. belly and sidepositions; reacted to painful irrita ion.

Cat Int-ravenous 0.5-5.0 5 Apathy, tottering gait, belly and sidepositions: greatly relaxed. Shortly after the injection, occasionaltwitching of the front extremities, salivation and vomiting.

Do 10.0-30.0 6 Short cramps, 5 minutes after the injection like above.With mg., death.

Dog Int-ravenous 0.5-2.5 3 Unsteady gait, slowing-down. Strongrelaxation, partly side position. None or weak reaction to apain-inducing irritation. Effects increase with the dosage. Occasionaltwitching of the front extremities. Salivation and vomiting.

Do 5. 0 2 As above; one animal developed cramps for a short time.

Do 10. 0 1 Shivering cramps, aggressive during about 5 minutes, thenlike above.

Intramuscu- 5. 0-15. 0 5 Atactic, side position, no reaction topain-inducing lar. irritations. Efiect increased with the dosage. Do 20.0 1 Intermittent cramp attacks; otherwise as above.

Por0ral 50. 0-150. 0 5 Tired to atactic till side position. Resultswidely variable and not always reliably dependent on the dosage. Do 200.0 1 Side position, later intermittent cramp (spasm) attacks, nextmorning dead.

Rhesus mondeyz. Subcutaneous- 0. 5 l Ptosis (drooping of the uppereyelid), sluggish movements, fatigue, limp extremities. 1. 0 2 Ptosis,sluggish movements, great fatigue, muscular relaxation. 2. 5 1 About aswith 0.5 and 1.0 mgJkg. 5.0 5 Ptosis, heat drooping, great fatigue,languid, pallor. 1-50 9 O.W. (without effect). Frog (Tempo- Into the100.0 5 After V hour, sluggish for 4 hours. Rear position ranch andlymph not tolerated. Esculenten). pouch. 200. 0 3 After houlr, rearposition tolerated; 1 animal died,

2 survive (2) Ataractic properties as developed in the hamster (Table2).[Literaturc: Krcislrott and Vatcr, Arch. f. expcr. Path. u.Pharmakol. 1959, vol. 236, p. 100.]

In this test a focused heat ray (beam) is directed upon the animalstail. As the analgesic effect, the prolongation of the reaction time(normally lasting 4 seconds) up As an emotional reaction in mammals thefright to 12 seconds is evaluated. The retraction of the tail positionassumed by the hamster was relied upon. Hamis considered to be thereaction to the pain. The De50 stcrs counteract against unfamiliar ordominant related is that dose, at which 50% of the animals are analgeticmates, adversaries, or in the case of a fright, with a. (insensitive topain) 20 animals for each docs. typical defense reaction. They throwthemselves on their Result.Mcdium effect. back, spread their extremitiesapart, and frequently utter (b) Tooth test, applied to rabbits (Table3).[Lita cry. Tranquilizing drugs affect the intensity of this eraiure:Hertle, Schanne and Stafe,Arzneimittelforschung reaction. 1957, vol. 7,p. 311.]

TABLE II In this case, the upper incisors of the animals are [Inhibitionof the gefer se rgacgoroi nl a gold hamster, irritated by increasingamounts of current during 10 Wh7286 if; 51;: 2O mscc., Hz. (measured inma.) till the animals show a Methaminodiazepoxide 10 defensive reactlon.The cur rcntstrength which with an chloropmmazine 30 untreated animaleventuatcs in said defensive movements, 1s called threshold value. The100 A-valuc is that dose B De'l'Q is meant that dose in man/ c, by whichthe of the investigated compound which is needed for raising 231 machosuppressed m of the ammals Started 50 the threshold value 100% asagainst the initial position.

IieSMZ'TStmDg g Eff t u th oil p i zsu fe sl, applied to cats (Table4).- sla ilisc fi giitig iisift iz s lezfiergi agbmsslon m eite;r5atur%: ;5 ]1dy. Jour- Pharmp py 1932.

D vo ,p. lzgLlratcggguie Abramson and Crans, Science 1954, vol. mil tipsof i slilueezed by mean; 9 a spring-pressure me er y W 1c t e pressureexerte 1s gg l g iig gg g f color adaptanon m the guppy i lnliiicatcd ink(g pm. Miasure of thclanalgdcsicdcifecfiz e pressure in g. sq. cm.increase to crac un ert e literature: Furst and Cuttmg Neumpsychopharma'action of the compound as against the initial value. Irricologl'a 1959308'] 69 tation reaction: Defensive movement of the animal ac- In the zg fig gg with F companied by vocal manifestations. The dose withtiveness towar s was surprislng see a c That this is also the case withcold-blooded animals was 23:55; g mcrease of 5 1- 18 J about shown bythe tests with fishes. A complete inhibition f .the ggressiveness of thefighting flSh OCCUI'IEd only 5 Far H1728? About 1 with 100 ,/1inhibition of the color adaptation of For mfjrphme About 2 mgjkgtheguppy aft r 50 mg./l, In a fighting fish Wh7286 For tE1101ental A110 7[Hg/ks has only in the guppy /s of the effect of chloro- Result.-Stron-geffect.

Promazine Methanigdiazepoxide is in a fighting" fish Since, in contrastwith morphine, Wh7286 has a powertwice as effective as Wh7286. 70 ful,central-attenuating effect on cats, too, it appeared op- (4) Analgesicproperties; portune to make a comparison with the analgesic proper- (a)Analgesia test with mice (mouse-tail test) Table ties of a narcotic. Inorder to preclude the real narcosis 3.-[Literature: Wolff, Hardy andGoodell, 1 our. Clin. effects, the analgesia was tested at the time ofthe recovery Investigations, 1940, vol. 19, p. 659-Wirth, Arch. Expcr.of the upright-standing capacity of the animals. The ref- Path. u.Pharmakol, 1952,vol. 216,1). 77.] crcnce compound used was thiopental(Table 5).-

r, Arologie,

h intra- 59503991 0 0 3 4-52 5 &

56070540 0 0 4 5 nmL5m guomandibular and on of the test com- DL50/A100paratus, partly by means of rs (Kymographion) ep spinal anaesthesiaWh7286 had mg./kg. (intravenous), which leads of the threshold valuebeyond the AP (kg/sq. cm.) at minutes after injection perimentellePathalogie und Pharmak intravenous 0052 451367 2 6 7 7 02 L&6 5 0 3 1 11With a cat under a de Result.Strong effect.

(b) Rabbits (Table 7).[Literature: Hoflmeiste On wide-awake, intactrabbits the variations of the Result-Strong eflect.

(6) Influence on the body temperature:

(a) Temperature drop in normothermal cats (Table Result.Distinct effect.

DL50 rabbit Rabbittooth intravenous test mg./kg.

strips on a Schwarz EEG ap mechanically-actuated scribe the same effect.

chiv fur ex threshold values of the licking and jaw-opening reflexarising in response to a dental irritation was determined for thereflexes in response to lin homolateral fiexures under the acti pound.That dose in to a 100%-increase initial position, is called the A100value.

8).-The action of the compound was tested wit venous administrationtwenty hours prior to the start of the test and during the entire testthe animals were held 1961, vol. 241, P. 178.]

5 in a thermostable room at 22 C.

Initial portion, k ./sq.cm.

DL50/DE50 DE50 mousetail test, mgJkg.

TABLE 4.-ANAL GESIA-CAT TAIL INTRAVENOUS INJECTION Average value fromanimals Initial portion, kg./sq. cm.

Average value from animals TABLE 3.ANALGESIA IN THE MOUSE AND RABBITDL50 mouse mg./kg., subcutaneous Dose, mgJkg.

515550005000 0 LL2 4 1 2 &7 0 5 0 112 Dose, mgJkg.

rch. of Inst. Pharm. 1947,

On flexing the in- Result.W-h thiopental.

Compound Codcin (base) Pethidine (base) Dextropropoxyphene-Phenoperidine Compound Morphine HCl Morphine, 0.2%

Thiopental, 2%

Compound Morphine, O.2%

Thiopental, 2%

(d) Analgesia test on the inflamed rat paw.[Literature: Randall andSelitte, A vol. III, p. 409.]

10 and 20 mg./kg. of Wh7286, subcutaneous, were ineflective.

(e) Rat joint test.[Literature: La Belle and Tislow lour. Pharmacol. andExperim. Therapy, 1960 p. 19.]

In this test, about 18 hours before the administration The absence ofthis reaction while the compound is acting, is evaluated, as theanalgesic effect.

The DESO of Wh7286 lies at 7 mg./ kg. (subcutaneous).

Result.Medium-high effect.

(5) Eflect on polysynaptic reflexes (central muscular relaxation).[Literature: Cardot, Laudier, COmptes-Rendus de la Societe de Biologie,1922, vol. 86, p. 529.]

[Sherington, Journal of Physiology, 1917, vol. 51, p. 404. Slater andLeary, Journal of Pharmacology, 1950, vol. 100.]

(a) Cats (Table 6 ).-The influence on the polysynaptic, homolaterealreflex and on the linguomandibular reof the compound to be tested 0.2ml. (cc.) of a 1% ver nitrate solution was injected into the rear ankleof rats. An acute arthritis sets in.

flamed joint by the experimentator the animals scream 55 with pain.

(b) Antipyresis (Table 9).Wh7286 was investigated flex, as well as onthe monosynaptic patellar reflex was inin doses of 0.1-5.0 mg./kg. withfebrile guinea pigs. vestigated on intact animals under a gentlechloralose- Resall.Distinct effect. urethan narcosis. The release tookplace every 5 seconds 7 O (7) Influence on barbiturate narcosis: by anelectrical irritation of the tongues tip, and, respec- (a) M0 use.Thehexabarbital narcosis of the mouse tively, of the Nervus tibialis. Thepatellar reflex was re- (100 mg./kg. of hexobarbital, subcutaneous) wasdetectleased (instigated) by an electromagnetically controlled ablyintensified by Wh7286; above 10 mg./kg. (s.c.) a hammer with a frequencyof l/sec. Every 3 nerve deepening of the narcosis extending to 2 stages(accordtwitches were recorded partly by means of extensometer ing toMagnus-Arn-dt and a. prolongation by 66 minutes In the latter Percentinhibition Within min. after the administration titative Patellar reflexsemi-quan respiration rate which can be Well AIOOwalue, mgJkg.(intravenous) (a) Rabbfts.The respiration volume of weak rabbits wasinvestigated by the Scholz experimental method (spirometer respirationinstance the animals exhale through a mask i to a gasometer. Control ofthe inhalation and exhalation is effected by Gildemeister valves.

5-10 mg./kg. of Wh7286 (intravenous) cause a slight slackening of thecounteracted by 5 mg./ kg. of Metrazol (i.v.).

Result.S1ight inhibition.

TABLE 5.ANALGESIA CAT TAIL estigated.

were observed, and with more than mg./kg. (s.c.) a deepening extendingto 3 stages, i.e., prolongation by minutes. The body temperature of theanimals was lowered by 10 and 20 rug/kg. (s.c.) 2 and 3 C.,respectively, as compared with the hexobarbital control test.

(b) Cat (Table 10).A corresponding action of Wh7286 on the thiopentalnarcosis, too, was inv Results.For (a) and (b): strong effect.

(8) Influence on respiration:

TABLE 7.REFLEXES OF RABBITS D ose, a/ intravenous n a s u 2 t mm50322O346348 fi5 700000107100O-m000 u 1.1 oc .6 ws m m 1 Men M m A e025050000 2 0 075 50525052 .00 m ,H C 328646630o emm%%2nd1 111M6644464 rn 0 he? m 1 1 1 w a m H I I mi am t wnm mu n 4 tims A x am h H SH D e et t h P es m .m e m h a w I t n e M 123123112311231.231231231234561234 Sw W 1433541319u221100022 in T a a M m F H 0 mm F 0 N O N S U 0 E B I O 500 U X X n rm UHMMG6 M%OM%BW4S%U 0 7 0 5 0 6 9. n 11 wl r 1 1 2 an t 0 mE 6 w M .1 6 13 5 .,.mm L r. M 8 A. u e F m i a 3% E m Pmw ma D R b .1 t.1 d n u 6 w .w 1533742522332211124 d n n E m m n L 0 t O u w B m m p nA m o m w B T L a 2 m. N 7 h .m W T %505005000000000 Compound 0 ompoundChl0rpr0mazin.

Methamin0diazepoxide- Phcnyraxnidol Mephenesin..

Carisoprodel.

(empty stomjccted 50 mg./kg. of tration of 0.2/1111. per

Hours after administration trode with 20 ma., 50 Hz., impulse durationof 10 msec. for 1 second.

Result.-Medium-high effect. (b) Metrazol cramp-Into each sober ach)mouse was intravenously in Metrazol, with a solution concen 20 grams ofthe animals wei ht within 4 seconds.

Result-Inefifcctiw.

tion after minutes .For this purpose 0.75 mg./kg. itrate was injectedintravenously. In all ited by tolto 3 mg./ kg.

other respects, as with (b).

TABLE11.THE EFFECT OF WH7286 ON THE RESPIRATORY 35 of strychnine n ADuration TABLE 8.TEMPERATURE D ROPCATS Maximum Normaliza, 20

drop, 0. (average values) HE THIOPEN'IAL NAR- A Stage LIMINARY ADMIN- 30th 1 & 8 &7 is .mnwfihm pfl oowwmoonoquwaonowm DMO n M da 1 3r. sm t121212121212 5 MM om n a n Mm u n u n n m u u n n n or t AYE mm I c v e.v v v m s 1 s .L .L i 5 5 m m 5 w 1% w m u u u u m n u w m .m m n m m 0u m C w I 6 p a 8 r b 9. O 0 F1 1 X 1 h e W C H FEVE R) Number ofanimals THE CAT BY THE PRE TABLE 9.ANTIPYRESIS ON FEB RILE GUINEA PIGSCompound mgJkg. (sub- COSIS OF ISTRATION OF WH7286.

TABLE 10.POTENTIATION OF T Wh7286, 10 mins.

Strychnine cramp, D E50, mg./kg.

Nicotine cramps, DE50, kg.

Electrically Cardiazole induced cramp, DE50, mgjkg.

TABLE NO. 12.ANTICONVULSION ACTIONMICE Mode of administration"Subcutaneous. Per os Per 0s.

Compound 1 Without effect. With oral administration shock after 3 hours.With subcutaneous administration shock after hour.

torn. Nephenesine Subcutaneous. Phenyramido1 d0..

(nullification) of the tonic spasm component was evaluated as an effect.

(a) Vertically-induced cramp (spasm).The animals were shocked across acranium electrode and a nose elec- Wh7286 Phenobarbital Diphenylhydan-Goesswald,

' compounds in rats.-The general aspect of poisoning by apomorphine,tryptamine and methamphetamine is based above all on the liberation ofbiogenic amines. In the following table the inhibition or the preventionof cramps (spasms) and stereotype reactions of the rat is considered toconstitute a measure for the action of the compounds.

TAB LE 13 by the preliminary subcutaneous administration of- Distinctsuppression of the poisoning in use p Wh7286, Chlorprorug/kg. mazine,rug/kg.

Tryptamine 40 mgJkg. intravenous 30 1O Apomorphine 1.25 mgJkg.intravenous- 20 40 Methamphetamine 10 rug/kg. intravenous- 2t) 25Result:

With tryptamine Wh7286 Clcdorpromazine With apomorphine Wh7286Ch1orpromazine With methamphetamine Wh7286 Chlorpromazine (l2)Electroencephalographic investigations:

(a) Cats.In a total of 8 animals, carrying electrodes implanted in thecranium, the influence of Wh7286 on the spontaneous-EEG, as Well as onthe reticularly, rhinencephalically and thelamically induced potentialswas measured. For details regarding the method and practice, seeHoffmeister, Psychopharmacologia, 1961, vol. 2, p. 27, and PflugersArchiv, 1961, vol. 273, p. 396.

(i) Spontaneous-EEG (10 tests); dosage: 1-5 mg./kg. i.v.Leading-off wasvia the frontal and occipital cortex, veutromedial thalamus, nucleuscaudatus, hippocampus (dorsal), temporal pole, and mesencephalicreticular formation.

Result-Within 1-10 minutes after the injection there occurs a distinctactivation of the spontaneous-EEG, which is distinguished particularlyin the cortex by a highfrequency low-voltage EEG, and in the temple-pole(thalamic) fibers of some animals by heaped-up 20-30 Hz. amygdalae. Withhigher doses (5 mg./kg.) there may during this time appear cramppotentials, such as separate spikes or short series of spikes-waves(possibly in rhythm measurable in seconds), which frequently spreadsynchronously with motoric tics (sporadic muscular contractions) intothe fore extremities. During this time the animals are already in theside position. In the further course there appear with majority ofcases, independently of the dosage, an increased number of 8-12 Hz.temporal pinions or to bursts of printed high-voltage waves which canfrequently be recognized synchronously in all the leading-oilconductors. Simultaneously therewith, the vigilance tonus (tonic spasm)in the tonsillar formation and in the hippocampus region decreases, ascan be recognized from the attenuation of the 30 Hz. pinions, or in thedisappearance of the 3-5 Hz. rhythm.

(ii) Induced potentials (Table 14).-Under the influence of Wh7286 (3mg./kg. intravenous), chlorpromazine (5-7 mg./kg., i.v.) morphine (5mg./kg., iv.) and hexobarbital (20 mg./kv., i.v.), administered to 3cats carrying chromically implanted electrodes, was comparatively testedfor potentials induced by intracerebral electrical irritations(stimuli), as well as for the EEG-vigilance reaction incited by a buzzersignal. There was thus evaluated (interpreted):

(1) Induced potentials, which can be led oil from the frontal cortex bya low-frequency electric stimulus of the formatio reticularismesenoephalica. The frequency of the stimuli was 5 HZ. Each separatestimulus is answered in the cortex by a potential variation of a specialform and amplitude.

(2) Recruiting potentials in the cortex, which were induced by anelectrical stimulus (5 Hz.) of the ventromedial thalamus.

In one animal, there was furthermore investigated the form of theafter-discharge in response to an electric stimulus applied to thetemporal pole, under the influence of the aforementioned compounds.

([1) Rabbits:

(i) Sp0ntane0us-EEG.-- In rabbits, there occurs under the influence ofWh7286 (1-5 rug/kg, intravenous) a distinct increase of the potential,accompanied by a decrease of the frequency, and by synchronization.

The typical theta rhythm in the hippocampus lead-off branch is blocked.The maximum effect appears about 10 minutes after the injection. Thevigilance reaction in response to an acoustical stimulus is generallysuppressed, that in response to a painful stimulus is readilyattenuated. After the administration of 10 mg./ kg. of Wh7286 (i.v.) ormore generalized shocks, which are accompanied by a high mobility,become predominant.

(ii) EEG-arousal, from intracerebral stimuli.The infiuence of thethreshold of the EEG-arousal reaction in response to intracerebralstimula was tested on rabbits, The initiation of the reaction waseffected by a highfrequency stimulus (250 Hz.) in the reticularismesencephalicus formation, in the ventral hypothamus, and in theventromedial thalamus. Stimulus threshold with a nontreated animal,about 0.1-0.5 volt for the reticularis stimulus, about 1-3 volts for thethalamus and hypothalamus stimuli. Threshold variation in A voltsmaximum; this means that, with every animal, the maximum threshold riseachieved within 60 minutes after the administration is, as compared withthe preliminary check test of the arousal reaction (in volts), each EEGvariation in the cortex or hippocampus lead-oil branches (as the casemay be) with the symptoms typical for the reaction (small amplitude,high frequency or synchronization, as the case may be).

Evaluation: Cortex arousal, at least 10 msec, and for the hippocampusarousal, at least 6 seconds per stimulus.

(iii) Antagonistic action against centrally active compounds in theEEG.The known EEG efiects of methamphetamine (5 mg./kg., i.v.) LSDmg./kg., i.v.), Levallorphan (l0 mg./kg., i.v.) in rabbits weredistinctly blocked by 2 rug/kg. i.v. of Wh7286; 5 mg./kg. i.v.suppressed an onset of nicotine spasms.

The EEG activation brought about by nicotine (0.5-3.0 mg./kg., i.v.)physostigmine (0.4 nag/kg, iv) and tryptamine (5 mg./kg., i.v.) could,however, not be broached by tolerable doses of Wh7286.

(13) Surgical tests (Table 15) .The efiects of Wh7286 described above(viz., stuporous condition, analgesia, re traction) made it seemreasonable to test the compound in experimental operations instead ofnarcotics.

TABLE 14 Chlorpromazine,

' mg./kg.l.v. Morphine, Hexobarbltal, Compound W]: 7286, 3 mg./kg. i.v.5 mgJkg. i.v. 20 mgJkg. m7.

Cat N 44 43 39 44 43 39 44 43 39 44 43 39 N0. of tests 1 1 2 1 1 1 l l 11 1 1 Response in the cortex tqanelectrwfllretmular (l) l (l) i il l l 1111 ill ill ill stunulus. 7 Recruiting T TTT TT 4 TTT T i ii iii llllUHil TT Tonsillar after discharge 1 l l T l l Vigilance reaction(Bussersigna.l)not ill ill ill ill TT TTT TTT ill ill ill tested.Permanent arousal Significance of symbols:

l 1 l =distinctly attenuated =0.b. (without outstanding eilect) 1 i=attenuated T =slightly stronger J, =slightly attenuated T t =stronger TT =distinctly stronger TABLE TABLE 15C'ontinued Animal Dosage, NumberAnimal Dosage, Number kind mg./kg., of Report kind mg./kg., of Reporti.v. animals i.v. animals Cat 5 1 Blood-drawing incision 0n the F 4 2-44 Intubation, successful with 2 aniextremity and subcutaneous incimals,th any partially, 1 sion without reaction, gentle repulfense. With 2animals, insertion sive movements on bluntly sepawas impossible due toresistance. rating t e muscular flesh, no 3 2 Repetition with theseanimals, after reaction during the skin suture. a week intubation wassuccessful. 6 (2 x 3) 1 Laparatomy, incisions into the skin andsubcutaneous tissues, with no reaction; piercing incision into mus- 1 5preliminary 5 x 2 within 60 minutes, then 5 Evipon-Na. cular tissues andperitoneum, with 2 Premedication: 0.1 mg./kg. (subcutaneous) ofSandolectil 30 minutes no defensive movements, likewise before. nopressing lifting out the colon, 3 Without premedication. and digitalprobing of the a dom 4 +3 thiopental 5 minutes later. rial cavity; 8Qminutes after the e d ResuZL-Distinot analgesia, particularly on theskin, pronounced mus- Of h P I p y to Stand oular relaxation, conscienceapparently maintained partly. Striking upright again restored.bloodlessiiess of the skin. 1 Trepanation, stereotactic insertion of i0Itlleep-saiaated elect)rodes, lan?r 2315- ac in y screws an eec o esocket to the cranial roof. Dura- P I edommantl) p p efiects tion of theoperation: 3% hrs. (14) I hibi i f d Normal course of the operation. Dog2 1 ,1 Lapapqtomy, incision m the Skin, (a) Oedema of the mts paw (Table6) .Due to thc fi i mm the Eentoneum d llljectlOll of bakers yeast(20%-suspension, 0.1 ml.) into on lifting out the cart and colon, noreactiomsuture m muscuiar the planta pedis of rats an oedema is formed.The intissiies, subcutaneous tissues and skin with no reaction;immediately tensity of said oedema (dropsy) is measurcd by means ofafter the operation the animal is the antiphlogmete-r WK -8 mentioned byKempcr and g g gf g appealablei Stand Ameln (Zeitschrift furcxpcrimentclle Medizin, 1959, vol. Dog i 2 1 Laparatomy: Incisions intothe skin, 131, p. 408) obtainable from the Gescllschaft fur Elcktrog ggfg iggggfifi ggifgg fifig physikalischcn Apparatebau Benshein Theadministrafiq and mcsenterlal root 110 tion of the test compounds tookplace 4 hours after the straining, abdominal integuments greatly mSutures home with oral administration of yeast, the testing of theantioedono reaction. Animal gets on its 7 feet immediately at the end ofthe matic effect every hour during the follow ng 3 hours. I ii);p%ra i0nand 1112151 sta c ti cally. l The inhibition of the oedema Was indicatedin percent of a s. Rhesus 1 a n i in i tgs sh e t he i ii jectiggj sidea preliminary test, i.e., calculated on the intensity of themollkeysugared; after. 10 oedema shortly before the administration ofthe test utes incision into the skin at the back; blunt palpitatign finesubcompounds or in scale divisions of the antiphlogmeter. out neoustissues an o e musg, Sheath with no repulsive RESMlL-DlStlllCt effect,somewhat 111cc that of predmmovements; Skm Suture, Wlth some. The sameeffect was obtained with adrcnalectomreaction. After 60 minutes, can sitdown, and after 170 minutes stands izcd animals; no antagonism ascompared with serotonin u right spontaneously.

2 1 Surgical care of a laceration (1 1 cm. is present m thl s test long,on the arm). 5 minu s (b) Formalin oedema of the rat (T able 17).-Inthis $55, 9 gfifi figffig iigi ii figfig instance the same procedure asstated under (a) Was m?- .About m t after adopted. Instead of yeast 0.1ml. ofa 3%-formalin soluthe IDJQOtlOl'l, the animals sit downspontaneously. Within the next 2 tion was lIlJCCtd into the plantapcdis. g fs hurt extremlty not Result.Distinct effect, about as withprednisone.

(c) Zymasan-oedema test, according to Lauenstein, Friedrich andHaberland, Med. exp., 1962, vol. 26, p. 200:

Result.Distinct effect (at least twice as strong as with Novocain).

TABLE Iii-INHIBITION OF A YEAST-INDUCED OEDEMA OF THE RATS PAW NumberDose Percent inhibition animals Maximum inhibition, in percent ofCompound Dose, mgJ N0. of the preliminary 2 mgJkg. per os 11. kg. per 05Animals control test, 8 mgJkg. per os 10 31. within 180 16 mgJkg. per os10 31 (Prednisone 29% 11111.). minutes 1 mgJkg. subcutaneous- 1O 2mg./kg. subcutaneous 10 22. 10 4 mgJkg. subcutaneousl 10 45 (Prednisone19% ink). Wh7286 2 29 8 mg./kg. subcutaneous- 10 42. 4 35 49 16 mgJkg.subcutaneous- 10 50 (Prednisone 21% inh.). 8 10 45 0 5 mgJkg. 1.1). 1016 44 1 o mgJkg. i.p 10 9. 31. 5 1o 38 2 0 mgJkg. to 10 17. 63 5 57 4 0mgJkg. in 10 22. 15 Prednisone 2 15 23 s o mgJkg. i 10 22. 4 25 47 16 0mgJkg 1 p 10 40 (Prednisone 28% inh.). 8 20 45 16 2o 46 31. 5 1o 41 e310 50 (d) Formalm-oedema test, accordzng to Domen oz and Phenylbutazone2 1o 33 'Theobald,,Exper., 1958, vol. 14, p. 33.-Dosage, as under 20 g28 23 (a), except that 0.1 ml. of a 3%-formalin solution was 16 15 56injected into the back paw of each animal. 5 i3 Amidozon 2 1O 25 3 hi itNumber Dose of Percent inhibition 25 5 g animals 63 1o 51 Ph 1 1 IN Cl 1m0 5 61 2 mg. /kg. per os 10 6. 0g a so s mgJkg. per 0s.-- 10 15. 65 1010 mgJkg. per os 10 22 (Prednisone 10% inh.).

30 TABLE l7.INHIBITION OF A FORMALIN OEDE'MA IN THE RAT PAW (e)Cotton-pellet test.Procedure: Two sterilized cotton pellets, weighing 10mg. were implanted below the Maximummhibition in percent of neck skin offemale albino rats under livipan narcosis. Compound Dosemgll N0- ofthprefimmmy The compound to be tested was administered per cs 7 5 kg.per 05 Animals control test, times at 24-hour time mtervals. On theeighth day the i g g animals were killed with chloroform, the cottonpellets removed and their dry weight determined. The inhibition M17286 45 32 was expressed in percent of a sodium chloride control i u s 5 25test. For each dose 10 animals and 20 cotton pellets, 40 16 5 37respectively, were used. Prednisone g g Dose/ day Percent inhibition 6 533 Big/kg- P 05 3 Phenylbutazone 4 5 3a 1 mg./kg. per os 17 g :2 2mg./kg. per os 11 (Prednisone 34% inhibition) Physiological N201 s 16 4mg./kg. per os 19 Recapizulatian.Wh'/286 has a distinct antiphlogisticTABLE 18.CUTANEOUS (EPIDERMAL) AESTHESIA eifect, above all in the Zymasetest, which is nearly in D ration f thee t're anaesthet'c agreemgnt wlththe results oijtamed W121} i yeist m Concentration Animal N0. COI IGitiOII $100 dalis ivith fine bnish duced oedema. In the essentiallymore stringent foreveryZmjnute5 NQv0cai11 malin-induced oedema test aneffect is llkewise detecta- M17286 ble. The effect is about of the orderof magnitude of prednisone. In the cotton-pellet test Wh7286 is only 01Mm 1 0 10 faintly efficacious. 2 0 12 (15) Local anaesthesia: 05% f; g32 (a) Conduction anaesthesia.-[Experimental procedure 1.0% 5 22 36according to Wirth, Deutsche Zahnarztliche Zeitschrift, g g3 $3 1960,vol. 15, p. 1270.] s 18 60 Into the rat tail were injected neutralsolutions of physiological NaCl (pH 7.1-7.3) containing 0.1%, 0.5% and1.0% Wh7286. The tail was then electrically irri- (16) Effect onsmooth-muscle organs: (a) Isolated guinea-pig intestine.By the Magnustated. With 0.1 Wh7286, there was no effect. With th d th followingvalues were obtained: 0.5%, complete conduction anaesthesia. After 3.2min- TABLE 19 utes, and with 1.0%, complete conduction anaesthesia Iafter 2.9 minutes. (Aver-age values of 5 animals per dose.) Inhibitionof Nicotine com Result.Strong effect (order of magnitude like thatCompound the acetylcho- BaCl -contractraction by of lidocaine/baycaine).ggifgjifg by W (0) Surface anaesthesia (Table 18).Brought about by I aneutral 0.1, 0.5, 1.0% solution in physiological NaCl 10 000 25409 50dropped into the connective membrane cyst near the I 0 909 4,000 rabbitscornea. For the purpose of comparison a pro- Pethidme iig'w 228 ,23caine solution having the same concentration was injected ifltO theother y Acetylcholine, 12.10 mach, 1110 Nicotine, 1:10.

Resalt.No particular anti-acetylcholine effect, strong anti-BaCl efiect,strong anti-nicotine effect.

(b) Isolated seminal vesicle (rat).-Th e contraction of the seminalvesicle (Samenblase) brought about by 2000 of Adrenalin/l. Was inhibited50% by 1000 of Wh7286. The corresponding value for chlorpromazine is107/1.

Result.No specific efiect.

Isolated uterus (rabbit).0n the isolated uterus of non-pregnant rabbitsconcentrations of 107 to 100 mg./l. have no specific efiect; 10 to 10mg./l., too, had no specific effect on the organ contracted by Adrenalin(1 mg./l.). Only at 50100 mg. of Wh7286 did a distinct inhibition of theAdrenalin-contraction become apparent.

(17) Anti-histamine efiect.In the experimental method according toSchaumann, viz. inhalation of an aqueous 0.15% histamine hydrochloridespray, by a guinea pig, 2.5 mg. Wh7286/kg, s.c., did not show anyanti-histamine effect.

18) Anti-asthma efiect.4ensibilization of guinea pigs by twointraperitoneal injections of chicken egg-white aqueous solution, 0.5ml. per dose). After 14 days inhalation of a 5%-aqueous egg-White spray.

By a preliminary administration of 1 mg. Wh7286/kg. (subcutaneous)slight protection; by 5 mg./kg. complete protection. Reduction of theintensity of the eggwhite asthma by about 50% eifected with 2.5 mg./kg.With these doses the animals were weak.

Result.Distinct anti-asthma effect duced).

(19) Effect on intestinal mobility (excretion of boneblack).-[Literature: Journal of Pharmacy and Pharmacology 1957, vol. 9,p. 380.]

In full-grown rats the passage through the small intestine of asuspension of bone black in a tragacanth mucilage was inhibited byWh7286 (1 mg./kg. per 0s), and 20% by 5 mg./kg., p.o.

(20) Diuresis.In the slightly modified experimental procedure accordingto Burn (cf. Wirth, Deutsche Medi- (eggwhite-inzinische Wochenschrift,1957, p. 1908), and with a total 22 of 20 rats, the excretion of urinewas slightly increased in the first six hours by 0.05 mmol of Wh7286/kg,p.o. (=11 mg./kg.). The excretion of Na+, K+, C1- and the titrationacidity of the urine were not significantly changed.

Resalt.Slightly increased water diuresis.

(21) Examination of the synaptic efiect:

(a) Mayoneural transmission-In the narcotized cat the contraction of themuse. tibialis ant. in response to an indirect irritation by Wh7286(1-10 mg./kg., i.v.) is not inhibited. Succinylcholine/iodide (0.2mg./kg., i.v.) prevents the contraction. The synaptic nerve-muscletransmission (relay) is thus not impaired by Wh7286.

(b) Nictitating membrane.In the narcotized cat, Wh7286 in doses of 1-2mg./kg., i.v. causes a distinct, long-lasting nictitating membranecontraction; it is not attenuated by the preliminary administration of aganglion blocking substance (Ecolid, 0.5 mg./kg., i.v.) or by theextirpation of the ganglion cervicale superius. The contraction of thenictitating membrane is intensified by Wh7286, whereas the contractionbrought about by arterenol remains unaffected. As is known,chlorpromazine (0.2 mg./kg., i.v.) diminishes the contraction of thenictitating membrane in the case of a preganglionar irritation of thesympaticus at the neck. The subsequent administration of Wh7286 (2mg./kg., i.v.) and preganglionar irritation causes a further lesseningof the nictitating membrane contraction; simultaneously therewith theblood-pressure-lowering action of chlorpromazine.

(22) Effect on the circulation:

(a) Dog and cat (vigilant) Table No. 2-0.A polyethylene tube wasimplanted into the Arteria carotis or femoralis of the animals underether or halothane narcosis. The administration of Wh7286 took placeonly after the animals had completely recovered from the narcosis.Measuremnts of the systolic and diastolic blood pressure, and of thepulse frequency by means of a Schwartz bloodpressure chamber; recordingeffected by means of a scriber. Wh7286 Was passed into the venajugularis or Was administered intramuscular.

TABLE N0. 20.-BEHAVIOR OF THE CIRCULATORY SYSTEM IN THE WIDE- AWAKE DOG.

Dog Weight, Sex Wh7286 Initial Behavior of the vascular No. kg. mgJkg.condition system under Wh7286 1 10. 0 Male 1 i.v BD 120/90 BD T 135/100in 5 minutes in min. up to 120/90 1 60 in 8 mins; stable P 1 up to endof test (90 min.) 2 9. 0 Female-- 3 1.v BD 130/90-.- BD T 180/125 P 90-P 1 60 in 10 min; constant 60 min; 30 after 4 hours.

3 8. 7 Female 3 iv. IBD /70.-. BD T160/130 in 10 min;

in 60 min. up to 1 /80;

/86 after 4 hours.

P70 P 1 40 in 15 mm; constant for 60 min; 45 after 4 hours.

4 10.5 Female.-- 5 i.v- BD 130l70 BD T 165/120 in 20 min;

1111 90 min. to 150/120; 120/90 after 4 hours P80 Pin 10min; 901'01 90min; 70 after 4 hours. 5 10. 5 Male 1 i.v BD 120/90--- BD T 200/ in 10min; in

min. to 120/60.

P 150 P 1 70 in 60 min. 6 I 9. 1 Male 3 i.v BD 120/90..- BD 1 250/190 in10 mins;

1in 120 min. to /110.

P 165- P180, at once constant.

1 Premedication: Atropine sulf. 0.05 mgJkg BD=B1ood pressure. P lse.

. l.v., 10 minutes later Wh7286. P: u

Doses of 1-3 mg./kg., i.v. caused in the dog a mediumhigh increase ofthe blood pressure which slowly fades away again after -60 minutes.Surprisingly, a distinct brady-cardia was observed.

Following the administration of atropine, the rise in blood pressure byWh7286 is substantially more distinct, and a drop of the pulse frequencybelow physiological values is in the present instance prevented to alarge extent.

With a total of 12 cats and Wh7286 doses of 1-5 mg./kg. basicallysimilar results were obtained. On repeated injection of the compoundinto the same animal the circulatory reactions became slightly weaker,although there was no pronounced tachyphylaxy.

Succinyl choline (1 mg./kg., i.v.), d-tubocurarine (0.2 mg./kg., i.v.),thiopental (up to 10 mg./kg., i.v.) do not substantially change theeiiect of Wh7286 on the circulation of dogs, once the blood-pressurefluctuations specific for each compound have died away.

(b) Narcotized cat (chloralose-urethane).(Blood pressure in the venafemoralis recorded via a Hg manometer.)

In doses of 0.3-2.0 mg./kg., i.v. Wh7286 causes an increase of themiddle blood pressure of 20-80 mm. Hg for 10 minutes, up to 2 hours. Inpart of the animals the blood-pressure rise was preceded by a morepronounced negative prevariation. In some cases there occurredirregularities during the blood-pressure rise.

By a preliminary administration of atropine (0.05 mg./kg., iv.) theblood-pressure reaction was slightly intensified, not inhibited byyohimbin (1.0 mg./kg., i.v.), Ecolid (0.5 mg./kg., iv.) andchlorpromazin (0.2-1.0 mg./kg., i.v.). On the blood-pressure reaction ofserotonin, tryptamin and noradrenalin, Wh7286 has no specifie effectAfter the extirpation of the carotis sinus follows a blood-pressuredrop, the nictitating membrane reaction is not altered.

(c) Spinal or beheaded cat.In an artificially breathing spinal cat(decapitation below the medulla oblongata) 2 mg./kg. Wh7286, i.v.,brought about a rise in blood pressure. After reaming out the spinalcord there occurred a rise in blood pressure likewise with thoseblood-pressure test objects that reacted well to adrenalin and arterenoladministered after 0.5-2 mg./kg., i.v.,

Wh7286. In the animals no longer possessing a brain and spinal cord, thepulse frequency was not impaired by the compound.

(d) Effect on the peripheral vascular vessels.(l) On the vascular systemof the frog (Rana esculenta) prepared according to -Lawes-Trendelenburg,200-500- 1000 Wh7286 had a slight vasoconstrictive effect.

(2) On the corresponding warm-blooded preparation obtained according toKochmann and Katel by flooding the lower half of a guinea-pigs trunk, adistinct lessening of the flow after SOD-1000a ensued in 6 of 9 tests.

(3) Finally, in a rabbits ear transfused (flooded) according toBissenski-Rischlieter there arose likewise a tendency to a slightvasoconstriction; still any dose-effect relationship was notdiscernable.

(4) In a narcotized dog, measurements of the rate of flow of the bloodthrough the arteria femoralis by means of the Vater fiowmeter gave thefollowing data:

TABLE 21 Minutes, after 2 mg. of Wh7286/kg., LV- Dog Start (1) bloodpressure 145/100 135/105 165/140 170/140 170/140 Amount of blood, ml/

min 29 9 1s 13 (2) Blood pressure 135/100 140/120 145/125 140/120130/110 Amount of blood, ml./

min 7 5.7 4.2 5.3 9

As will be seen, the decrease of the peripheral flow of blood is notindependent of the pressure.

According to Langendord, 0.1-100 Wh7286 have on the isolated guinea pigheart no specific effect as regards the amplitude and rate of flowthrough the coronary artery; with higher doses diminution of bothamplitude and rate of flow have been established. No influence onfrequency.

(b) Oxygen content of venous coronary-sinus blood. In order to obtain anindication as to the effect on the coronary the O content of the venouscoronary-sinus blood of the intact narcotized dog under the influence ofWh7286 was determined by means of a catherization of the heart.

In the same manner as in the non-narcotized animal these tests showed anincrease of the systolic and diastatic blood pressure, the amplitude ofthe latter decreased slightly.

(0) Efiect on the electr0cardi0gram.-On enfeebled trained dogs the EKG(bipolar standard lead-oil branches, I, II and III) was taken up underthe influence of 1-3 mg./kg., iv. and 5-10 mg./kg. intramuscular bymeans of subcutaneous needle electrodes. Recording was done on aSchwarzer multiple-channel direct scriber. With all animals thereappeared shortly after the administration of the compound a pronouncedbradycardia (abnormally slow heat-action) with emphatic respiratoryarrhythrny. The latter may have been due to the strong sedation of thedogs.

In 2-16 administrations on 13 animals there occurred, about 10-40 m.after the administration of 1-3 mg./kg. Wh7286, short-timedinterspersions of extra ventricular systoles. In view of the frequencyof heart-rhythm anomalies in non-narcotized dogs this finding is of noparticular importance. Said rhythm anomalies are, besides, frequentlyobserved in man and dog in cases of peripheral sympathicomimetica.

(24) Efiort on the blood gases.-On vigilant dogs the O and CO contentsof arterial blood were determined by the Van Slyke apparatus before andafter the administration of 1-3 mg./kg. Wh7286, i.v., every half hourover a period of time of -120 minutes. No change in the O and COcontents.

III. Compatibility 1 Distribution according to Miller and Tainter.Proceedings of the Society of Experimental Biology and Medicine, 1944,vol. 57, p. 261.

E Kg. MgJkg.

Weight of animal: practically no change.

Blood picture: red blood picture, no deviation; in the white bloodpicture of I, perceptible shift to the left and slightly higher BSR;(II) and (III) o.b.B.

Urine: with II, after the 11th and 15th injection slight turbidity dueto albumin, a few erythrocites in the sediment; with III only a trace ofalbumin after the 11th injection (III) inconspicuous.

Residual nitrogen: with (II), rise above the initial value after the thinjection, again normal after the th injection; (II) and (III) o.b.B.

Liver activity: BSPand thymol values o.b.B. On the day after the lastadministration, the animals treated with Evipan-Na were dead.

Dissection:

(I) Heart o.b.B., lung o.b.B., weight 9 g./kg.; liver congested; leftkidney capsule, difficult to detach; right kidney, remaining abdominalorgans, brain, o.b.B.

Histological observations: abcess in the left renal cortex, straitedinfiltrations and connective-tissue bands from the cortex into theinterior of the kidney (secondary evidence), in several tubuli, cellulardebris and albumin. Liver, spleen, had very much blood, otherwiseo.b.B., adrenal gland o.b.B.

(II) Heart, o.b.B., lung o.b.B., weight 8.2 g./kg.; liver, spleen,highly congested; remaining abdominal organs,

brain, o.b.B.

Biological observations: kidneys, epithelial debris in several tubules;spleen highly hyperaemic; adrenal glands, o.b.B.

TAB LE 24 Group I Group II Group III (controls) At the start 63 61 68 6468 63 After 42 days 227 166 233 162 216 176 After 98 days 344 210 334211 338 218 After 161 days 404 244 378 248 403 240 Blood: in the red andwhite blood picture, no deviations from the control tests.

Urine: in group III (controls), occassional traces of albumin, in thesediment laminar epithelia bacteria and phosphates; pH 6.1 to 7.3.

In Group II, more frequently traces of albumin; pH 6.1 to 6.6, otherwiseas in the controls.

Group I, same as the controls; pH 6.1 to 6.4.

Further experimental evidence. Group I:

1 d dead after 7 days 1 9 killed after 94 days, ailing Group II:

2 6 dead after 10 and 18 days, resp.

2 9 dead after 41 and 143 days, resp. Group III:

1 d dead after 42 days (control) The other animals show nothingnoteworthy.

After 100 administrations, 2 male and 2 female animals of each groupwere killed with ether: Dissection: macroscopic, no essentialdifferences between controls and the Wh7286 animals.

Microscopic, numerous desquamated epithelial tissues (III) Heart,o.b.B.; lung, o.b.B.; weight, 10 g./kg.; old scars on the surface of theleft kidney; capsule, detached with difficulty; right kidney, remainingabdominal organs, brain, o.b.B.

Histological evidence: liver and spleen, congested; epithelial debrisand albumin in several tubules of the left kidney.

Recapitulation.No essential changes to be attributed to Wh7286.

(b) Rats.-Three groups, each comprising 10 young male and 10 femaleanimals. Initial weight 60-70 grams.

Group (I) 10 mg./ kg. in the Altromin feed.

Group (II) mg./ kg. in the Altromin feed.

Group (III): Altromin-control doses daily on 170 consecutive days.

The weight changes of the animals treated with Wh7286 deviated butlittle from the control data:

(b) Marginal vein, 0.1 mil, blocked for 1 minute, rabbit ear:

TABLE 26 1. o.b.B 1. o.b.B 1. o.b.B. 2. o.b.B 2. 0.10.3 2. Slightinflammation, after 2 days o. 3. Slight inflamma- 3. Slightinflammation, after 2 days o.b.B.

tion, after 2 days o.b.B.

3. Slight inflammation.

(c) Intracutameous, 0.1 ml, rabbit ear, in a 1-5% concentration causes alocally limited inflammatory infiltrate.

(d) Intracutarteous, 0.1 1111., on the forearm: of three experimentalpersons:

0.1% and 0.5% solution (base) in a physiological NaCl 27 solution,adjusted with HCl to pH 7.0 and 5.0, resp. Each swelling (lump) caused'by 0.1 ml. (cc.). EX- cept for the puncture injection was painless;extent of swelling and vicinity (location of the dermal lymph crevices)anaemic; in this region contraction of the erectorus pilorum (goosepimples).

Anaesthesia in the swelling area during 1540 minutes with the 0.1%solution, and about 60 minutes with the 0.5% solution. Thereafter nolocal irritating effect (as in the controls with the NaCl solution).

(28) Additional tests with humans.The compound (weight predetermined)was dissolved in about 50 cc.

of water (slightly acid) and taken in the early forenoon hours.

TAB LE 27.OB SE RVATIONS Dose, mg./ Test Person 1 Test Person II personper s 2 After min. slows down somewhat. After 1 hour, o.B.

"- Somewhat tired after the first hour.

15 After 30 minutes, tired,

pronounced sleepiness, after 4 hours 0.13.13.

During the first hour,

slowly developing lassitude till about 2 hrs. after the administration.

After 3 hours, the lassitnde subsides somewhat; after 6% hrs.. o.b.B. 40After mm., sluggish In the first hour, slight lassitude becoming slowlygreater. Heavy eyelids, pallor oi the face, after 90 minutes slightburning of the eyes. After 1% hrs. very strong lassitude; falls asleepin the ab sence of external stimuli. Slight uncertainty on walking.Lassitude no longer so pronounced; after 2 hrs. only slight, after 6hrs. During the following hours slowing down of the spontaneous andvoluntary reactions. After 12 hrs. fell asleep several times whilequietly active (reading). Pallor has subsided. After 14 hours (evening)fell asleep quickly,

deep sleep. Woke up the usual time (22 hrs. after the administrationo.B.). Electrocardiogram, o.b.B. bloodpressure check test andtemperature were within normal limits.

heavy lids and limbs, in the further course, sleepiness, after 1 hr.some atactic dryness of the month. In the absence of fairly strongexternal stimuli, falls asleep. After 2 hours, slow subsidence of theeffect; after about 5 hrs., subjectively o.B. During the activity of thecompound, pronounced paleness oi the skin. Electrocardiagram (EKG)0.b.B. Blood pressure control and. temperature were within normallimits.

IV. Discussion and recapitulation:

As demonstrated by the foregoing data, W=h7286 is a compound endowedwith versatile pharmacological properties. The activity spectrumcomp-rises ataract-ic, analgetic, tranquiliz'ing, antipyretic,antiphlogistic, antic-onvulsive, locally panesthetic, and antiasthmaticelfects, in various degrees.

Standing in the forefront of the central eitects is a strongataractive-sedative component. The latter was particularly marked in thehamster, cat, dog, monkey, andas shown by a few tests conducted withpersons in man. Little senstive were the mouse, rat, frog and fish. Itis possible that as regards the sensitiveness to Wh72-86 there existrelationships with the degree of development of the brain, as is wellknown with the case of hypnotics and anticonvulsive compounds.

' The strong effect of analgesic compounds on the rabbit and cat-in thiscase less than in the rat and mouse-arose above all in the stages ofataraxia and sedation. Distinct qualitative relationships exist betweenthese eifects. In evaluating analgesia tests as conducted with animalsit should always be borne in mind that one is here concerned withactions on reflex mechanisms,

and that these tests do not reveal any unconditioned testimonial poweras regards the becoming conscious of pain." With this reservation,Wh7|286 is to be included in the group of the more intensely eflicaciousvanalgetics. It is noteworthy that the breath depressing efiect which isparticularly disturbing felt with some analgetics, is relatively lowwith Wh72'86 (rabbit, dog). The spastic diverse inhibitionoften typicalfor analgesics which can be readily detected in the rat, is totallyabsent with Wh72l86. Conversely, a certain degree of inhibition of theintestinal mobility is observed in the rat, possibly in connection withthe distinct musculotrop-spasmolytic ac tion.

In the spontaneous EEG of the cat, Wh7286 can be diflerentiated fromanalgesics of the morphine-type and from barbiturates such aslhexobarbital, also as regards its influence on recruiting potentials,although in the latter respect there is a parallel relationship withchlorpromazine.

The slight reticulo-cortical attenuation present in the EEG and thedistinct thalamo-cortical activation do not explain sufiiciently theunusually strong centrally depressing properties of the compound. It isprobable that part of its main action lies in the inhibition of spinaland bulbar reflex mechanisms. Beyond this, it is conceivable that, underthe influence of Wh7286 there occurs a change in the central aifercncesnot detectable by the EEG methods used in the present investigations. Inthe cortex and cerebrum of cats and rats the serotonfine level indicateda certain tendency to drop after Wh7 286 had been administered in theheretofore conducted tests; the catecholamine a tendency to rise.However, these changes are not essentially significant. In this respectthe compounds effects are like those of chlorpromazine, with which ithas several other central properties in common, as, for instance, beingpartly stronger than chlorpromazine, the antagonistic effect evidencedin opposition to the central excitation by tryptamin, apomorphine andmethaniphetine in the rat, which for methamphetamine could also bedetected in the EEG of the rabbit. The EEG activation by nicotine in therabbit is not influenced by Wh7286 and chlorpromazine. However, thereare also considerable differences between the two compounds.

In the isolated guinea-pig intestine, Wh7286 exerts a strong nicotineaction, in contrast with chlorpromazine.

Again, in contrast with chlorpromazine, Wh7286 lacks to a far-reachingdegree the cataleptic eifect (rat) and the antematic efiect (d-Ogtreated with apomorphine). On the contrary, with the dog, vomitingoccurred at the higher subcutaneous doses. In the monkey and in the testpersons, no vomiting was observed.

For certain purposes as an analgesic the staracticstuporous action ofthe compound may become a disturbing factor. For others it is perhapsnecessary, aboveall in conjunction with the relaxation eifect: with thecat, dog and monkey it was possible, following an intravenousadministration to proceed with surgical operations while consciousnessappeared to be maintained to a far-reaching extent. Incisions into theskin were well tolerated. In those instances where the compound alonewas insufficient, it became possible to proceed with small additionalbarbiturate doses.

Although the compound has in non-excessive doses no specific efiect onthe heart and the coronary transfusion, the intravenous administrationis accompanied by a transient tonic action on the circulation. Thisaction occurs in the narcotized and non-narcotized intact animal (dog,cat), in the spinal (decapitated) cat and, finally, after extracting thespinal cord, hence is essentially not of a central origin. Obviously,Wh7286 causes the peripheral sympathico tonus (bodily tone) to increase.This is in concordance with the powerful contraction of thesympathetically stimulated nictitating membrane of the cat when thecompound is injected intravenously.

The nictitating membrane contraction, as well as the rise in bloodpressure, due to Wh7286, occurs also after a preliminary treatment withEcolid R (a ganglion-blocking compound) as after the expiration of theganglion cervicale superius, hence is not the consequence of ganglionicstimulation, at least not in the tested dosage range. The traditionalvascular-test animals, viz. frog and rabbit ear, afforded weakerfindings than was expected; by flooding the lower half of the rabbittrunk the vasoconstriction was more distinct, particularly, however, inthe rear extremity of the dog. One surprising finding isvasoconstriction on the hand in the case of intracutaneous orsubcutaneous injection in man (selftreatment). It is to be assumed thatthe pallor of the face in man and monkeys after an oral or intravenousadministration is likewise the consequence of any such vascularreaction.

The rise in blood pressure is accompanied by a lowering of the pulsefrequency. In the intact vigilant and narcotized animal (dog, cat) thebradycardia (abnormally slow heart action) is prevented by atropine, butwith a simultaneous increase of the blood-pressure rise. The reductionof the pulse frequency can be interpreted as a counter-regulatoryexcitation (stimulation) of the vagus nerve center across the pressorreceptors. This view is also supported by the reversal of theblood-pressure action by Wh7286 after the expiration of the carotissinus, which apparently leads to a predominance of the vagus(depressor).

As shown by the tests conducted by the investigators on their ownbodies, the ataractic-sedative effect outlasts considerably the tonic(invigorating) effect on the blood pressure. During the at-rest positionthe blood pressure drops below the initial values with reascent to thenormal value during the gradual vanishing of the ataraxia and sedation.

Also noteworthy is the strong local-anaesthetic effect; the antitussiveeffect is probably connected therewith in some way, which obviously isalso promoted by the sedative action of the compound.

The inhibition of the oedema by Wh7286 is considerable. In the case ofthe oedema caused by yeast in the rats paw, the adrenektomised animalswere found to be not less sensitive than the normal ones. As regards theevaluation of the oedema tests it should be noted that their specificityis dubious. Positive findings in the test with animals do not justifythe assumption that a genuine antiphologistic effect is now to beimplicitly found in man.

The toxicity of the compound corresponds to its strong pharmacologicalaction. From the hitherto conducted tests with su bchronicadministration to dogs days) and chronic administration to cats (170days), it can be inferred that the compounds do not cause deep-seatedpathological changes in the organs. The tolerance as regards thevascular and muscular systems was sulficiently high in animal tests.After the intracutaneous injection into rabbits, damage to tissues(lesions) were observed; in man, 0.5% solutions, injectedintracutaneously, were found to be still tolerable, causing a stronglocal anaesthesia. In tests conducted by the investigators on their ownpersons the oral administration was unaccompanied by incompatibilitysymptoms.

V. Suggestions as regards indication and dosage:

In is indicated that Wh7286 will possess clinical utility for thefollowing:

1) In psychiatry for the treatment of neurosthenic psychoses.

(2) In internal medicine and surgery, as an analgesic in cases of greatpain.

(3) As an analgesic in cases of specifically indicated arthritis(indication With a view to the relaxating and antiphalgistic actioncomponent).

(4) Internal medicine; as a hypnotic. (5) In anaesthesiology forpremedication and as an independent anaesthetic, as the case may be.

SUGGESTED DOSAGES Re (1): Oral, single dose; parenteral, single dose-2040 mg.; up to ing/day 10 to 20 mg. (max.) intramuscular or slowly i.v.within 5 minutes, up to twice a day.

Re (2): Oral, single dose; parenteral, single dose- 20-40 mg; up tomg./day 10-20 mg./(max.) intramuscular, or slowly intravenously within 5minutes up to 3 times a day.

Re (3): Oral, single dosel020 mg. up to 3 times a day.

Re (4): Oral, single dose20-40 mg. in the evening.

Premedication.-Parenteral, single dose lO-2550 mg. slowly intravenous.Becomes effective after a short latency (about 5 minutes after theinjection). Caution: potentially narcotic! As an independentanaesthetic.Dosage based on experience with premedication.

All the dosage data relate to adults. As to the difference between thesexes nothing is known concerning them up to the present. Caution is tobe exercised from the viewpoint of the commercial security factor inview of the strong ataraxia and sedation.

In the case of overdosage, it is advisable, according to the extentstate of the investigations, to use Pervitin subsequent to the action.

Modes 0 dispensation as a drug:

(a) Tablets, up to 20 mg, glazed, with indented groove.

(b) Capsules, 1% aqueous solution, capacity 2 to 5 ml.

What is claimed is:

1. A chemical compound selected from the group consisting of compoundsrepresented by the formula:

wherein R is a member selected from the group consisting of di-loweralkylphenyl-; tri-lower alkoxyphenyl-; lower alkyl-chlorophenyl-;tri-lower alkylphenyl-; dichlorophyl-; trichlorophenyl; andpentachlorophenyl-; and acid addition salts of the same with non-toxicinorganic and organic acids.

2. The chemical compound,2-(2,6-dimethylphenylamino)-4H-5,6-dihydro-1,3-thiazine.

3. The chemical compound, 2 (2 ethyl 6-methylphenylamino)4H-5,6-dihydro-1,3-thiazine.

4. The chemical compound, 2 (2 chloro 6methylphenylamino)4I-I-5,6-dihydr0-1,3-thiazine.

5. The chemical compound, 2- (2,6 dichlorophenylamino)-4H-5,6-dihydro-1,3-thiazine.

References Cited by the Examiner Frankel, Chemische Berichte, vol. 30,pp. 2509 (1897).

Tisler, Archiv der Pharmazie, vol. 293, pp. 621-626 (1960).

WALTER A. MODANCE, Primary Examiner. NICHOLAS S. RIZZO, Examiner.

1. A CHEMICAL COMPOUND SELECTED FROM THE GROUP CONSISTING OF COMPOUNDSREPRESENTED BY THE FORMULA: